Biologically active oils

ABSTRACT

A process for the production of fats or oils and their extracts containing biologically active chemical compounds from a lipid substrate, the process comprising: a) inoculation of a lipid substrate with fungally derived lipolytic enzymes; b) incubating the inoculated substrate for a period of between about 7-120 days at a temperature of between about 435° C., at a humidity of between about 75-100%, and c) processing said substrate mixture to obtain a biologically active fat or oil.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/559,599, filed Dec. 2, 2005, which was national stage filing under 35U.S.C. 371 of PCT/AU2004/000745, filed Jun. 4, 2004, which InternationalApplication was published by the International Bureau in English on Dec.16, 2004, which claims priority to Australian Patent ApplicationAU-2003902823, filed Jun. 4, 2003, each of which is hereby incorporatedin its entirety by reference herein.

TECHNICAL FIELD

The present invention relates to fungal metabolism/transformation oflipid substrates to produce fats and more particularly oils and theirextracts containing biologically-active chemical compounds for thetreatment or prophylaxis of diseases, disorders or conditions in humansand other animals.

BACKGROUND OF THE INVENTION

With the growth of the alternative/complementary medicine industry andthe general perception in some areas that the products are “snake oil”remedies, there is a pressing need to ensure that products supplied bythis alternative industry have reproducible efficacy and are safe foruse by the general public. However, due to recent adverse publicity bythe press and government regulators there is an absolute requirement foralternative medicines to meet strict guidelines regarding the quality ofthe raw materials and the method of manufacture. One of the majordifficulties with alternative medicines is that the ingredients used areoften composed of materials which in most cases may contain manydifferent chemical compounds. Hence there is an enormous challengeinvolved to ensure efficacy and safety of the products for use bysociety. One of the major reasons for the lack of knowledge of theeffectiveness of these products is that the alternative andcomplementary medical industry generally pride themselves on the factthese products are not animal tested, hence their claims cannot bevalidated.

Historically, the animal and plant oil industries are among the oldestin the world, hence procedures used in this industry are wellestablished. In addition the industrial and medical applications, ofwhich there are many, are well documented in the literature. However,many oils such as olive, evening primrose oil, flaxseed oil, cod liveroil and emu oil are used to treat a variety of medical conditions anddiseases but virtually no attention has been paid to the difficulty inreproducing efficacy and safety data for these types of products. Infact, over the past thirteen years of animal and chemical testing of alarge variety of many different types and batches of animal and plantoils, large variation in biological activity and chemical compositionhas been observed.

It has been increasingly recognised in this area that in order toincrease the credibility of complementary/alternative medicines based onanimal and plant extracts, particularly oils, quality control in termsof reproducibility of efficacy and safety data for these types ofproducts is of foremost concern.

OBJECT OF THE INVENTION

It is therefore an object of the present invention to provide processesfor the production of animal and plant-derived oils and their extractscontaining biologicallyactive chemical compounds having therapeutic andprophylactic activity in respect of a wide range of diseases andconditions in humans and other animals.

It is another object of the present invention to provide pharmaceuticalcompositions formulated for administration by any route, includingwithout limitation, oral, buccal, sublingual, rectal, parenteral,topical, inhalational, injectable and transdermal, preferably oral ortopical, including biologically-active oils and/or their extracts(therapeutic oils and extracts thereof) which demonstrate efficacyacross a broad spectrum of diseases, disorders or conditions in humansand other animals, together with pharmaceutically acceptable excipients,carriers or adjuvants.

It is also an object of the present invention to provide a method forthe treatment or prophylaxis of a wide range of diseases, disorders andconditions in humans and other animals by the administration ofbiologically-active oils and/or their extracts obtained according to theprocesses of the present invention.

It is also an object of the present invention to provide a method forthe treatment or prophylaxis of a wide range of diseases, disorders andconditions in humans and other animals by the administration of thefatty acid esters and amides of biologically-active oils and/or theirextracts.

It is a further object of the present invention to provide the use ofbiologically-active oils and/or their extracts in the manufacture ofpharmaceutical formulations for the treatment or prophylaxis of a widerange of diseases and conditions in humans and other animals.

SUMMARY OF THE INVENTION

This invention is based on the identification of the major factor/sresponsible for the variation in quality of plant and animal derivedoils, namely the transformation of the lipid substrates by fungi growingin and on the lipid substrate. The lipid substrates used are obtainedfrom animal and bird fat (obtained by cutting/slicing of fat tissue),plant tissue, seeds, nuts etc. appropriately treated (by either cutting,crushing etc). The quality of the fat or oil produced by this solidstate process depends on the type and number of fungi, lipid substrate,temperature, humidity and length (time) of incubation prior torendering, cold pressing, solvent extraction or supercritical fluidextraction. The basis of this invention therefore relates to thecontrolled use of fungi to transform lipid substrate/s to produce oilswhich contain biologically-active chemical compounds by the solid stateprocess/s. It has been established by the present inventor that exposureof the lipid substrate to fungi is responsible for the major variationin biological activity (efficacy) of different therapeutic oils ratherthan temperature, oxygen and light during processing and storage,although these latter factors may have some minor influence.

Accordingly, a first aspect of the present invention provides a processfor the production of fats or oils and their extracts containingbiologically-active chemical compounds from a lipid substrate, theprocess comprising:

a) Inoculation of a lipid substrate with a fungal mixture,b) Incubating the inoculated substrate for a period of between about7-120 days at a temperature of between about 4-35° C., at a humidity ofbetween about 75-100%, andc) Processing, said incubated substrate mixture to obtain a biologicallyactive fat or oil.

Fats or oils containing different biologically active chemical compoundsmay also be produced by simply incubating the prepared animal or plantlipid substrate which is either sliced, ground, minced or chopped andperforming steps 2-3 above with careful adjustments to temperature, timeand humidity.

A related aspect of the present invention provides a solid state processfor the production of oils and their extracts containingbiologically-active chemical compounds from a lipid substrate, theprocess comprising

a) Inoculation of a lipid substrate with a fungal mixture havingenzymatic activity, said fungal mixture being derived from saidsubstrate,b) Incubating the inoculated substrate for a period of between about7-120 days at a temperature of between about 4-35° C., at a humidity ofbetween about 75-100%, andc) Processing said substrate mixture to obtain a biologically active fator oil.

In step b) typically the period of incubation is between about 7 to 56days at a temperature between about 5-20° C. and at a high humidity ofbetween about 80-100%.

In step c), if the lipid substrate is animal derived, it is typical thatthe product is a biologically active oil obtained by rendering saidinoculated substrate. Alternatively, if said lipid substrate is plant orseed derived, the biologically active oil is obtained by cold pressing,solvent extraction or supercritical fluid extraction of said inoculatedsubstrate mixture.

There is also provided a biologically active product, particularly anoil when produced by the process of the first aspect of the presentinvention described above.

The typical steps/procedures which must be performed in order to producethe biologically-active oils and fats; assuming we have pure cultures ofdifferent strains of fungi in storage are:

1. Inoculation of the sterilised lipid substrate (e.g. animal or plantlipid) with the appropriate fungal mixture;2. Incubating the above mixture for a specified temperature range,period of time and humidity;3. Rendering the above mixture;4. Centrifuging the rendered mixture produced in step 3;5. Filtering the oil produced from the centrifuge step;6. Sterilising the oil at 135° C. for two hours;7. Filtering the oil after sterilising;8. Storage of the oil;9. Extraction of the oil if required.

Providing the above procedures are repeated the oil produced will bereproducible within experimental error, as this is a biological processin which some variation will be certain to occur.

The above oils or fats can also be produced by simple preparing theanimal or plant lipid source by either slicing, grinding, mincing orchopping and then placing on polymer or stainless steel trays with orwithout perforations depending on the requirements and then carrying outsteps 2-9 above. This step simply involves adjusting the temperature,humidity and time for the metabolism/transformation of the lipidsubstrate to occur.

Bacteria are not involved in the transformation process. While notwishing to be bound by any theory, it appears that the fungi penetratethe lipid cell walls and secrete enzymes that transform/metabolise thelipid within the cell. Some metabolic products are released from thelipid cell into the medium and while some others are absorbed by thefungi and further transformed internally and either stored there orsecreted from the cells.

The fungal mixture used to inoculate the lipid substrate and which isthe source of the fungally-derived lipolytic enzymes may be intact/wholefungal organisms, pure fungi, single fungi or mixed fungi, active enzymeextracts thereof, genetically modified organisms or modified enzymes.The process of the present invention transforms the lipids substrate, tofats or oils containing biologically-active chemical compounds which aresuitable for treating a wide range of human and veterinary diseases. Itis noted that using this method of producing oils and their extracts theamount of free fatty acids, mono and diglycerides are enriched. Forexample the level of total free fatty acids in some animal and plantoils is increased to or exceeds 14%. The compounds found in the oils andtheir extracts act synergistically to give the desired biochemicalaction in humans and animals for the treatment of a wide range ofdiseases and conditions.

Typically, the lipid substrate can be selected from animal or plantsources, of either terrestrial or marine origin, or the substrate can beconstituted from lipids or their extracts impregnated onto artificialsubstrates supplemented with mineral and organic amendments (see Walleret al 2002, Plant Pathologists Pocketbook 3rd Edition, CABI, New York).Sources of animal lipids include goanna, sheep, chicken, emu, ostrich,camel, duck, geese, pig, cattle, horse, mutton bird, sea cucumber, fishand shellfish including mussels. Sources of plant oils includeseeds/nuts of macadamia sp, Canarium spp, peanut, sunflower, safflower,linseed, soybean, wheat, oats, barley, almond, avocado, cashew,quandong, maize, wattle, olive, palm and rice. Othervegetable/plant/seed sources include coconut, pili nut, ngali nut,tamanu nut, neem seed, sesame and canola.

Typically, the fungi inoculated onto the lipid substrate will have beenisolated from the substrate (ie is endogenous to the lipid substrate)and found to transform the specific or similar substrates found withinthe host lipid substrate. Such fungi are typically found in the sexualand asexual states of the Phyla Zygomycotina, Ascomycotina andBasidiomycotina. This is therefore understood to cover the fungiDeuteromycetes which is the asexual state of the main phyla. Fungi alsotypically found to transform lipid substrates include Phoma sp,Cladosporium sp, Rhodotorula mucilaginosa, Cryptococcus albidus,Trichosporon pullulans, Mucor spp, Epicoccom purpurescens, Rhizopusstolonifer, Penicillium chrysogenum, Nigrospora sphaerica, Chaetomiumglobosum. Fungi demonstrating the ability to transform or having thispotential may be typically improved by either traditional or moleculargenetic techniques to alter the fungi genotype.

The substrate may also or instead be inoculated with enzymes derivedfrom fungi. Typically these enzymes are endogenous to the fungi, but canalso be developed from alternative sources or genetically modifiedisolates. Such enzymes may also be purified and their activity increasedby alteration of their structure using physical, chemical, molecular orother techniques.

Typically, the lipid substrate is sterilised and then inoculated withone or more fungi or their enzymes as required. The substrate may besliced, minced, chopped or ground to enable it to be spread in a layertypically between 0.5 and 10 cm on a surface that may be a stainlesssteel tray, or with the base perforated to allow oxygen to the lowersurface and oil to drip from the substrate and be collected in asuitable container or an equivalent system. Inoculation uses standardprocedures (see Waller et al 2002) including spraying or painting thelipid surface with fungal spores suspended in sterile water. Thesubstrate is then typically incubated for a period between about 7 daysand about 120 days, more typically between about 7 and 63 days, or about7 and 56 days, or about 7 and 42 days, or about 7 and 35 days. Even moretypically, the substrate is incubated for a period between about 7 and28 days or about 7 and 21 days and most typically between 14, 21, 28,35, 42, 56, and 63 days. The inoculated lipid substrate is incubated ata temperature between 4-35° C., typically around 5-20° C., and arelative humidity between 80-100%, typically 95%.

Following incubation the animal lipid substrate including thetransformed or metabolised substrate, containing the fungi is minced orground and transferred to a stainless vessel prior to the renderingprocess. This process typically involves rendering at a temperaturebetween 40-80° C., typically around 70-75° C. with constant slow speedstirring until the lipid substrate has melted into oil. This oil is thentypically centrifuged and filtered and can be further extracted. Thefiltrate which contains the biologically active oil is then typicallyfurther sterilised by heating for a period of between about 15 minutesand about 8 hours, more typically between about 1 hour and 6 hours, evenmore typically between about 1 hour and 4 hours. Typically the filtrateis heated to a temperature between about 100-160° C., more typicallybetween about 110-150° C. and even more typically between about 120-140°C., most typically about 130-135° C.

In the case of plant seed, nut oils or other lipid sources not of animalorigin, the inoculated and incubated lipid substrate/s containing fungimay be minced or ground prior to cold pressing using a screw press, theoil from the screw press then being centrifuged and filtered. This oilis then typically sterilised by heating for a period of between about 15minutes and about 8 hours, more typically between about 1 hour and 6hours, even more typically between about 1 hour and 4 hours. Typicallythe oil is heated to a temperature between about 100-160° C., moretypically between about 110-150° C. and even more typically betweenabout 120-140° C., most typically about 130-135° C. The oil produced isthen treated as for animal substrates.

The oil obtained from the above processes may then be subjected tosolvent extraction which typically involves mixing the oil on a mass orvolume basis in the ratio of 1:1 or 2:1 solvent to oil then cooling at atemperature of between 0° C. to −40° C. for a time period from 30minutes up to 24 hours, typically 0° C. for 16 hours. The solvent isdecanted or poured off, centrifuged if required containing, andevaporated dryness to obtain the extract. The resulting residue containsthe biologically-active chemical compounds.

The oil or extracts from the above processes may also be subjected(including any derivatives obtained by chemical treatment of oils andtheir extracts) to the following processes in order to obtain specificfractions or chemical compounds,

(1) High performance liquid chromatography, experimental conditions usedwill depend on the chemical and physical properties of chemicalcompounds required for treatment of specific human and animal diseasesand conditions.(2) Super fluid chromatography, experimental conditions used will dependon the chemical and physical properties of chemical compounds requiredfor treatment of specific human and animal diseases and conditions.(3) Wiped-Film Molecular Still or Evaporator (eg Pope), experimentalconditions used will depend on the chemical and physical properties ofchemical compounds required for treatment of specific human and animaldiseases and conditions.(4) Hybrid Still incorporating Wiped-Film Evaporator and FractionalDistillation Column.(5) Molecular Distillation Plant or any combination of the above itemsin (3), (4) and (5), experimental conditions used will depend on thechemical and physical properties of chemical compounds required fortreatment of specific human and animal diseases and conditions.

A second aspect of the present invention provides a method for thetreatment or prophylaxis of a wide range of diseases, disorders andconditions in humans and other animals by the administration of thefatty acid esters and/or amides of biologically-active oils and/or theirextracts (therapeutic oils and extracts thereof). Esters include methyl,ethyl, propyl and isopropyl groups. Methyl and isopropyl esters of theextracts of these oils have been in-vivo tested successfully on rats forthe treatment of arthritis with success.

A related aspect of the present invention provides the use of the fattyacid esters and/or amides of biologically-active oils produced accordingto the present invention, and/or their extracts, for the preparation ofa medicament for the treatment or prophylaxis of a wide range ofdiseases, disorders and conditions in humans and other animals by theadministration.

Typically, the biologically active oils and their extracts (therapeuticoils and extracts thereof) produced can be used to treat and/or preventa wide range of diseases, disorders or conditions in humans and otheranimals. Typical diseases, disorders or conditions which may be treatedor prevented include: respiratory diseases or conditions such as asthma,bronchial disease and chronic obstructive pulmonary disease (COPD),vascular diseases or conditions such as atherosclerosis, coronary arterydiseases, hypertension and sickle cell disease-associatedvaso-occlusion, skin diseases or conditions such as various dermatitis,psoriasis and atopic eczema, all types of burns, gastrointestinaldiseases or conditions such as ulcers, gastric reflux, inflammatorybowel disease, ulcerative colitis, Crohn's disease, pancreatitis andperiodontal disease, cancers including bowel cancer and prostate cancer,sarcoidosis, septic shock, musculo-skeletal diseases or conditions suchas arthritis including osteoarthritis and rheumatoid arthritis, chronicjoint and ligament pain, leukemia, diabetes, allergy including otitismedia and ocular allergy, uveitis, dysmenorrhoea, kidney diseases orconditions including glomerulonephritis and nephritic syndrome andprostate diseases or conditions such as benign prostate hyperplasia, anda wide variety of inflammatory disorders. The biologically active oilsproduced can also increase bone mass density and improve bone strengthand connective tissue disorders.

The biologically active oils and their extracts (therapeutic oils andextracts thereof) appear to reduce the levels of C reactive protein(CRP) in the blood of animals and humans (Refer to test results forpatient 7 in the examples of therapeutic activity below). Hence theseoils and their extracts may used to treat a wide range of human andanimal diseases and conditions associated with an elevation of CRP.

CRP belongs to the pentraxin family of proteins, so-called because ithas five identical subunits, encoded by a single gene on chromosome 1,which associate to form a stable disc-like pentameric structure. It wasso named because it reacts with the somatic C polysaccharide ofStreptococcus pneumoniae, and was first discovered in 1930 by Tillet andFrances. In the presence of calcium, CRP specifically binds tophosphocholine moieties. This gives CRP a host-defensive role, asphosphocholine is found in microbial polysaccharides (where CRP-bindingactivates the classical complement pathway and opsonises ligands forphagocytosis), in platelet-activating factor (PAF) (which isneutralised), and in polymorphs (causing down-regulation).

CRP is exclusively made in the liver and is secreted in increasedamounts within 6 hours of an acute inflammatory stimulus. The plasmalevel can double at least every 8 hours, reaching a peak after about 50hours. After effective treatment or removal of the inflammatorystimulus, levels can fall almost as rapidly, as CRP has a plasmahalf-life of the 5-7-hours. Some of the most common conditionsassociated with major elevations of CRP levels are:

(a) Inflammatory diseases such as various forms of arthritis includingrheumatoid arthritis, psoriatic arthritis and juvenile chronicarthritis, Crohn's disease, ulcerative colitis, Reiter's disease etc.(b) Malignancies such as lymphoma, sarcoma.(c) Necrosis such as myocardial infarction, tumour embolisation andacute pancreatitis.(d) Trauma such as burns and fractures.(e) Rheumatic fever, tuberculosis, allograft rejection and leukemia.

In addition, the biologically active oils and their extracts(therapeutic oils and extracts thereof) inhibit the secretion ofprostaglandin PGE₂ from the mouse fibroblast cell line (see Table 9which summarises the Percent Inhibition of Secreted PGE₂ from 3T3 cellsexposed to Oil Extracts). This data demonstrates that these oils andtheir extracts appear to inhibit the cyclooxygenase pathways. Hencethese oils and their extracts can be used to treat a wide range of humanand animal diseases and condition associated with increases in the COXpathway activity. These types of inhibitors may be used to treatdiseases and conditions in humans and animals such as rheumatoidarthritis, osteoarthritis, pain, etc.

The biologically active extracts inhibit leukotriene synthesis byinhibition of the lipoxygenase pathways (including the 5-, 12- and15-LOX pathways.

The biologically active oils produced have also been noted tosynergistically enhance the efficacy of a variety of pharmaceuticalsincluding the corticosteroids, dexamethasone and prednisolone. Suchsynergy is of great clinical benefit as raised levels ofpro-inflammatory leukotrienes or 5-lipoxygenase are associated not onlywith asthma but also with rheumatoid arthritis, osteoarthritis,scleroderma and inflammatory bowel diseases such as Crohn's disease, andthe administration of an active oil of the present invention allowslower doses of steroids to be used. (See FIG. 4 for the synergisticaction of a lipoxygenase inhibitor plus a steroid.)

A third aspect of the present invention provides a pharmaceuticalcomposition comprising a biologically active oil, or a fatty acid esterand/or amide of a biologically-active oil and/or an extract thereof,together with a pharmaceutically acceptable carrier, excipient oradjuvant. Typically, the compositions can be in the form of immediaterelease, extended release, pulse release, variable release, controlledrelease, timed release, sustained release, delayed release, long acting,and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred form of the present invention will now be described by wayof example with reference to the accompanying drawings wherein:

FIG. 1 is a flow chart of the process for extraction of biologicallyactive oils from the crude oil/filtrate.

FIG. 2 is a flow chart of the process for production of biologicallyactive oils according to the present invention.

FIG. 3 is a summary of the enzymatic pathways acted upon by thebiologically active oils to suppress pro-inflammatory leukotrienes

FIG. 4 shows how steroids and the lipoxygenase inhibitor(s) (LI) from abiologically-active emu oil (therapeutic oils) can act co-operatively tosuppress pro-inflammatory leukotrienes.

FIGS. 5A-P show chromatograms, numbered 1 to 32, which reflect thebiological activity of each sample in Tables 1 to 8, as summarized inTable 11.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As is clear from the foregoing, this invention relates to the fungalmetabolism/transformation of lipid substrates. The fungi can beendogenous or exogenous to the lipid substrate. However, only fungi withvery specific capabilities will grow on lipid substrates. Not all havethe potential to transform the lipid substrate from which they wereisolated, or other lipid substrates. Hence the present invention canutilise all fungi which grow on and metabolise/transform lipidsubstrates to produce oils which contain biologically-active chemicalcompounds.

a) Isolation of fungi

Fungi are isolated and cultured from lipids using standard methods (seeWaller et al 2002 for some common methods). Fungi can be found growingon or near the surface of naturally colonised lipidic substrates.Typically, the fungi are isolated from lipid substrates naturallycolonised by the fungi and cultured onto standard agar media usingstandard techniques. Typically, fungi growing on the surface of lipidsubstrates are obtained by removing fragments of fungus with a sterileprobe and placing the fragment on media for subsequent growth andidentification. Typically endogenous fungi are isolated and culturedfollowing surface sterilisation of the lipid substrate with alcohol orhypochlorite. Fragments of the lipid substrate are then placed on mediacontaining antibiotics to suppress bacterial growth. Emerging fungi aresub-cultured to fresh sterile media for growth and subsequentidentification. Media typically include Potato Dextrose agar, MaltExtract agar, V8 juice agar, Cornmeal agar, Oatmeal agar. Typically, thefungi can be subcultured from isolation plates on the same or otherstandard media.

Such fungi can undergo traditional or genetic modification to enhanceformation and activity of target compounds. Fungi isolated fromdifferent lipid substrates can be tested for their ability to transformdifferent lipid substrates. Those fungi found to transform mosteffectively can replace less effective isolates to increase productionof biologically-active compounds. These processes are used widely toenhance production of biologically active compounds and were used, forinstance, to enhance production of penicillin. Enzymes can be isolatedand purified from the fungi and their activity maximised using physicalor chemical treatment techniques, according to R. K. Saxena, AnitaSheoran, Bhoopander Giri, W. Sheba Davidson, (2003) Review ofPurification Strategies for Microbial Lipases, 52, 1-18 which isincorporated herein by reference. Enzymes are only isolated from fungiwhich are known to grow on and transform/metabolise lipid substrates toproduce oil containing biologically-active chemical compounds.

b) Storage of Fungi and Enzymes

Fungi are stored using standard techniques such as lyophilisation,storage at low temperatures, in sterile water, on nutrient agar underoil, or desiccated (see Waller et al 2002 for some standards techniquesof storing fungi).

c) Storage of Lipid Substrates

Animal and plant lipid substrates are stored in enclosed containers.Animal lipids are stored at temperatures typically below −18° C. Animallipid substrates may be freeze-dried and ground or minced prior tostorage at low temperatures. Plant lipid substrates are stored understandard conditions of low humidity, typically below 9%, and temperaturetypically below 30° C.

d) Sterilisation of Animal and Plant Lipid Substrates (of Marine andTerrestrial Origin) Prior to Inoculation

Prior to transformation, either animal or plant lipid substrate issterilised in order to remove both endogenous and exogenous microbes.Sterilisation may include washing in ethanol or hypochlorite solution,gamma irradiation or its equivalent, or heat treatment (see Waller et al2002, Plant Pathologists Pocketbook 3^(rd) Edition, CABI, New York forexamples of techniques in common use). In addition, lipid substrate/smay be modified by addition of specific mineral and organic additivessuch as found in Czapek Dox agar (see Waller for a recipe for CzapekDox, a commonly used mineral supplement).

e) Inoculation of Lipid Substrate

Depending on the fungi population of the lipid substrate it isfrequently possible to simply metabolise/transform the lipid substrateusing the conditions specified below, without further inoculation of thesubstrate.

The lipid substrate is inoculated with one or more fungi or theirenzymes as required. The substrate may be sliced, minced, chopped orground to enable it to be spread in a layer typically between 0.5 and 10cm on a surface that may be a stainless steel trays, or with the baseperforated to allow oxygen to the lower surface and oil to drip from thesubstrate and be collected in a suitable container or an equivalentsystem. Inoculation uses standard procedures (see Waller et al 2002)including spraying or painting the lipid surface with fungal sporessuspended in sterile water. The inoculated lipid substrate is incubatedat a temperature between 4-35° C. commonly around 5-20° C., and arelative humidity between 80-100%, typically 95%. The substrate is thentypically incubated for a period between 7 days and about 120 days, andtypically between 7, 14, 28, 35, 42, 56, 63 days.

f) Production of Oils and Extraction of Biologically Active Compounds

The lipid substrate used during fungal metabolism will determine themethod used to extract the biologically active oil. The four mainmethods that may be used are: temperature rendering, supercritical fluidextraction, solvent extraction and cold pressing. The last method isused only in respect of the production of plant oils.

Following incubation the animal lipid substrate containing the fungi isminced or ground and transferred to a stainless vessel prior to therendering process. This process typically involves rendering at atemperature between 40-80° C., usual temperature set around 70-75° C.with constant slow speed stirring until the lipid substrate has meltedinto oil; heating may be electrical, or by steam or hot water. Theliquid in the vessel that contains the oil is then centrifuged, followedby filtration. The residue that remains from the centrifuging step maythen be subjected to further extractions, generally using standardprocedures used in the plant-seed oil and pharmaceutical industries aswell as in natural product isolations used in research. The filtrate,which contains the biologically active oil, is then heated for a furtherperiod of between 15 minutes and about 8 hours at temperatures rangingfrom 100 to about 160° C. under inert gas atmosphere or at normalatmospheric conditions. Typical conditions that are commonly used are135° C. for 2 hours under inert gas atmosphere such as nitrogen. Thisstep sterilises the oil, and in addition denatures any protein/spresent. After cooling to a suitable temperature using a heat exchangerthe oil is then filtered again to remove any residual precipitatedprotein and/or fungi particulate which may be present. The oil ispackaged into 20 and or 200 litre pharmaceutical grade drums forstorage. For extraction of biologically active compounds from oil referto FIG. 1.

In the case of plant seed, nut oils or other lipid sources not of animalorigin the inoculated and incubated lipid substrate/s containing fungimay be minced or ground prior to cold pressing using a screw press, theoil from the screw press then being centrifuged and filtered. This oilis heated for a further period of between 15 minutes and about 8 hoursat temperatures ranging from 100 to about 160° C. under inert gasatmosphere or at normal atmospheric conditions. Typical conditionscommonly used are 135° C. for 2 hours under inert gas atmosphere such asnitrogen. This step sterilises the oil, and in addition denatures anyprotein/s that may be present. After cooling to a suitable temperatureusing a heat exchanger the oil is filtered again to remove any residualprecipitated protein and/or fungi particulate. The oil is packaged into20 and or 200 litre pharmaceutical grade drums for storage. The cakefrom the press is solvent extracted using a range of common solventsselected from such as hexane, isohexane, petroleum spirits, methanol,isopropanol, propanol, ethanol and diethyl ether. The solvent is thenremoved by evaporation and recovered for future use. The techniques usedare standard in plant oil industry. The oil produced is then treated asdescribed for animal substrates.

The oil obtained from the above processes may then be subjected tosolvent extraction at various temperatures typically using one or moreof the following solvents: methanol, ethanol, propanol, isopropanol,diethyl ether, light petroleum spirits, butanol, acetone andacetonitrile. This procedure involve mixing the oil on a mass or volumebasis in the ratio of 1/1 or 2/1 solvent to oil then cooling to atemperature of between 0° C. to −40° C. for a time period from 30minutes up to 24 hours. The solvent containing the biologically-activemolecules is decanted or poured off, centrifuged (if required) andevaporated dryness to obtain the extract. Typical conditions on alaboratory scale 100 gm of oil is thoroughly mixed with 100 gm methanoland held at 0° C. for 16 hours, then centrifuged if required and solventevaporated using a rotary film evaporator. The resulting residuecontains the biologically-active chemical compounds. Solvent should berecovered for recycling.

Forms of Administration:

It is possible in the pharmaceutical composition of the inventivesubject matter for the dosage form to combine various forms of release,which include without limitation, immediate release, extended release,pulse release, variable release, controlled release, timed release,sustained release, delayed release, long acting, and combinationsthereof. The ability to obtain immediate release, extended release,pulse release, variable release, controlled release, timed release,sustained release, delayed release, long acting characteristics andcombinations thereof is performed using well known procedures andtechniques available to the ordinary artisan. Each of these specifictechniques or procedures for obtaining the release characteristics iswell known to those persons skilled in the art. As used herein, a“controlled release form” means any form having at least one componentformulated for controlled release. As used herein, “immediate releaseform” means any form having at least some of its pharmaceutically activecomponents formulated for immediate release.

A variety of administration routes are available and the route selectedwill depend on the particular condition being treated and the dosagerequired for therapeutic efficacy. In the methods and compositions ofthe present invention, any mode of administration is acceptable andinclude oral, rectal, topical, nasal, transdermal or parenteral (egsubcutaneous, intramuscular and intravenous) routes.

Any biologically-acceptable dosage form, and combinations thereof, arecontemplated by the inventive subject matter. Examples of such dosageforms include, without limitation, chewable tablets, quick dissolvetablets, effervescent tablets, reconstitutable powders, elixirs,liquids, solutions, suspensions, emulsions, tablets, multi-layertablets, bi-layer tablets, capsules, soft gelatin capsules, lard gelatincapsules, caplets, lozenges, chewable lozenges, beads, powders,granules, particles, microparticles, dispersible granules, cachets,douches, suppositories, creams, lotions, topicals, inhalants, aerosolinhalants, patches, particle inhalants, implants, depot implants,ingestibles, injectables, infusions, functional foods and combinationsthereof. The preparation of the above dosage is well known to thosepersons skilled in the art. Generally, each would contain apredetermined amount of the active component in association with acarrier which constitutes one or more appropriate ingredients.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active component which isformulated according to known methods using suitable dispersing andsuspending agents. A sterile injectable preparation may be formulated asa solution or suspension in a non-toxic parenterally acceptable diluentor solvent (eg water, isotonic sodium chloride solution). Sterile fixedoils can also be employed as a solvent or suspending medium.

Typical dosages:—(a) extracts (from 0.01 mg to 1000 mg per kilogram)several doses taken orally may be necessary throughout the day, (b) oil(from 5 mL to 20 mL per day) taken orally in 5 mL doses. Initial loadingdoses of up to 30 or 40 ml of pure oil per day is also typical. Multipledaily doses are contemplated to achieve appropriate systemic levels ofthe active component. The formulation of therapeutic compositions iswell known to persons skilled in this field. Suitable pharmaceuticallyacceptable carriers and/or diluents include any and all conventionalsolvents, dispersion media, fillers, solid carriers, aqueous solutions,coatings, isotonic and absorption delaying agents and the like. Suchformulations and formulating is described in Remingtons's PharmaceuticalSciences (18^(th) Edn), Mack Publishing CO, Pennsylvania, USA.

EXAMPLES A) Preparation of Topical Medicaments Using Biologically-ActiveOils

Examples of creams for topical application were prepared according tothe following protocols:

Formulation 1 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 52.475Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.350 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 30.000 Prolipid 141 ™ Emulsifierblend of stearic acid, 5.000 behenyl alcohol, glycerol- monostearate,lecithin, C12-C16 alcohols and palmitic acid Cerasynt 840 ™ PEG-20stearate 1.000 Ceraphyl 230 ™ Di-isopropyl adipate 4.000 Vitamin Eacetate Vitamin E acetate 0.200 PHASE C 10% w/v NaOH Sodium hydroxide0.875 Water Water 2.000 PHASE D Liquapar Optima ™ Phenoxyethanol,methylparaben, 1.000 Isopropylparaben, isobutylparaben butylparabenTotal 100.000 MANUFACTURING PROCEDURE 1. In phase A combine water,disodium EDTA, and glycerol, then mix thoroughly. 2. Sprinkle StabilezeQM ™ into the pre-mixed solution with stirring at room temperature,until uniformly dispersed. Then heat to 75-80° C. while stirring, for atleast 30 minutes. 3. In a separate vessel, combine ingredients of phaseB; mix and heat to 75-80° C. 4. Add phase B to phase A, ,homogenise for3-5 minutes, then turn off the heat source. 5. Add phase C intohomogenate of phases A & B and then homogenise for 3-5 minutes with nofurther heating. 6. Remove homogeniser and thoroughly mix, while coolingto 38-40° C. 7. Add phase D to batch and mix until uniform. 8. Adjustfor water loss and mix until uniform. NOTES (a) Adjustments to theamounts of preservative may have to be made after challenge testing. (b)Small adjustments may have to be made to the Stabileze QM ™concentration to give correct viscosity depending on the application.(c) Small adjustments in composition will have to be made, to allow forfragrance if required. (d) Stabileze QM, Cerasynt 840, Ceraphyl 230,Prolipid 141 and Liquapar Optima are trade marks of InternationalSpecialty Products, Inc. 1361 Alps Road Wayne NJ 07470

Formulation 2 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 51.950Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.500 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 25.000 Neem seed oil Neem seed oil4.000 Tea tree oil Tea tree oil 1.000 Prolipid 141 ™ Emulsifier blend ofstearic acid, 5.000 behenyl alcohol, glycerol- monostearate, lecithin,C12-C16 alcohols and palmitic acid Cerasynt 840 ™ PEG-20 stearate 1.000Ceraphyl 230 ™ Di-isopropyl adipate 4.000 Vitamin E acetate Vitamin Eacetate 0.200 PHASE C 10% w/v NaOH 1.250 Water 2.000 PHASE D LiquaparOptima ™ Phenoxyethanol, methylparaben, 1.000 Isopropylparaben,isobutylparaben butylparaben Total 100.000 MANUFACTURING PROCEDURE 9. Inphase A combine water, disodium EDTA and glycerol, then mix thoroughly.10. Sprinkle Stabileze QM ™ into the pre-mixed solution with stirring atroom temperature, until uniformly dispersed. Then heat to 75-80° C.while stirring, for at least 30 minutes. 11. In a separate vessel,combine ingredients of phase B; mix and heat to 75-80° C. 12. Add phaseB to phase A, homogenise for 3-5 minutes, then turn off the heat source.13. Add phase C into homogenate of phases A & B and then homogenise for3-5 minutes with no further heating. 14. Remove homogeniser andthoroughly mix, while cooling to 38-40° C. 15. Add phase D to batch andmix until uniform. 16. Adjust for water loss and mix until uniform.NOTES (a) Adjustments to preservative may have to be made afterchallenge testing. (b) Small adjustments may have to be made to theStabileze QM ™ concentration to give correct viscosity depending on theapplication. (c) Small adjustments in composition will have to be made,to allow for fragrance if required. (d) Stabileze QM, Cerasynt 840,Ceraphyl 230, Prolipid 141 and Liquapar Optima are trade marks ofInternational Specialty Products, Inc. 1361 Alps Road Wayne NJ 07470

Formulation 3. COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water46.425 Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100Stabileze QM ™ Poly(methyl vinyl ether/maleic 0.650 anhydride) decadienecrosspolymer PHASE B Oil Biologically-active oil 25.000 Neem seed oilNeem seed oil 5.000 Cerasynt 945 ™ Glyceryl stearate & Laureth 23 6.000Cerasynt 840 ™ PEG-20 stearate 4.000 Ceraphyl 230 ™ Di-isopropyl adipate5.000 Vitamin E acetate Vitamin E acetate 0.200 PHASE C 10% w/v NaOHSodium hydroxide 1.625 Water Water 2.000 PHASE D Liquapar Optima ™Phenoxyethanol, methylparaben, 1.000 Isopropylparaben, isobutylparabenbutylparaben Total 100.000 MANUFACTURING PROCEDURE 17. In phase Acombine water, disodium EDTA and glycerol, then mix thoroughly. 18.Sprinkle Stabileze QM ™ into the pre-mixed solution with stirring atroom temperature, until uniformly dispersed. Then heat to 75-80° C.while stirring, for at least 30 minutes. 19. In a separate vessel,combine ingredients of phase B; mix and heat to 75-80° C. 20. Add phaseB to phase A, homogenise for 15 minutes, then turn off the heat source.21. Add phase C into homogenate of phases A & B and then homogenise for3-5 minutes without further heating. 22. Remove homogeniser andthoroughly mix, while cooling to 38-40° C. 23. Add phase D to batch andmix until uniform. 24. Adjust for water loss and mix until uniform.NOTES (a) Adjustments to the amount of preservative may have to be madeafter challenge testing. (b) Small adjustments may have to be made tothe Stabileze QM ™ concentration to give correct viscosity depending onthe application. (c) Small adjustments in composition will have to bemade, to allow for fragrance if required. (d) Stabileze QM, Cerasynt945, Cerasynt 840, Ceraphyl 230, Prolipid 141 and Liquapar Optima aretrade marks of International Specialty Products, Inc. 1361 Alps RoadWayne NJ 07470

Formulation 4 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 51.475Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.350 anhydride) decadiene crosspolymerPHASE B Oil Biologically active oil 25.000 Neem seed oil Neem seed oil4.000 Tea tree oil Tea tree oil 1.000 Prolipid 141 ™ Emulsifier blend ofstearic 5.000 acid, behenyl alcohol, glycerol- monostearate, lecithin,C12-C16 alcohols and palmitic acid Cerasynt 840 ™ PEG-20 stearate 1.000Ceraphyl 230 ™ Di-isopropyl adipate 4.000 Vitamin E acetate Vitamin Eacetate 0.200 PHASE C 10% w/v NaOH Sodium hydroxide 0.875 Water Water2.000 PHASE D Liquapar Optima ™ Phenoxyethanol, methylparaben, 1.000Isopropylparaben, isobutylparaben butylparaben Total 100.000MANUFACTURING PROCEDURE 25. In phase A combine water, disodium EDTA andglycerol, then mix thoroughly. 26. Sprinkle Stabileze QM ™ into thepre-mixed solution with stirring at room temperature, until uniformlydispersed. Then heat to 75-80° C. while stirring, for at least 30minutes. 27. In a separate vessel, combine ingredients of phase B; mixand heat to 75-80° C. 28. Add phase B to phase A, homogenise for 3-5minutes, then turn off the heat source. 29. Add phase C into homogenateof phases A & B and then homogenise for 3-5 minutes with no furtherheating. 30. Remove homogeniser and thoroughly mix, while cooling to38-40° C. 31. Add phase D to batch and mix until uniform. 32. Adjust forwater loss and mix until uniform. NOTES (a) Adjustments to preservativemay have to be made after challenge testing. (b) Small adjustments mayhave to be made to the Stabileze QM ™ concentration to give correctviscosity depending on the application. (c) Small adjustments incomposition will have to be made, to allow for fragrance if required.(d) Stabileze QM, Cerasynt 840, Ceraphyl 230, Prolipid 141 and LiquaparOptima are trade marks of International Specialty Products, Inc. 1361Alps Road Wayne NJ 07470

Formulation 5 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 51.475Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.350 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 25.000 Neem seed oil Neem seed oil4.000 Tea tree oil Tea tree oil 1.000 Prolipid 141 ™ Emulsifier blend ofstearic 5.000 acid, behenyl alcohol, glycerol- monostearate, lecithin,C12-C16 alcohols and palmitic acid Cerasynt 840 ™ PEG-20 stearate 1.000Eucalyptus oil Eucalyptus oil 4.000 Vitamin E acetate Vitamin E acetate0.200 PHASE C 10% w/v NaOH Sodium hydroxide 0.875 Water Water 2.000PHASE D Liquapar Optima ™ Phenoxyethanol, methylparaben, 1.000Isopropylparaben, isobutylparaben butylparaben Total 100.000MANUFACTURING PROCEDURE 33. In phase A combine water, disodium EDTA andglycerol, then mix thoroughly. 34. Sprinkle Stabileze QM ™ into thepre-mixed solution with stirring at room temperature, until uniformlydispersed, then heat to 75-80° C. while stirring, for at least 30minutes. 35. In a separate vessel, combine ingredients of phase B; mixand heat to 75-80° C. 36. Add phase B to phase A, homogenise for 3-5minutes, then turn off the heat source. 37. Add phase C into homogenateof phases A & B and then homogenise for 3-5 minutes with no furtherheating. 38. Remove homogeniser and thoroughly mix, while cooling to38-40° C. 39. Add phase D to batch and mix until uniform. 40. Adjust forwater loss and mix until uniform. NOTES (a) Adjustments to preservativemay have to be made after challenge testing. (b) Small adjustments mayhave to be made to the Stabileze QM ™ concentration to give correctviscosity depending on the application. (c) Small adjustments incomposition will have to be made, to allow for fragrance if required.(d) Stabileze QM, Cerasynt 840, Ceraphyl 230, Prolipid 141 and LiquaparOptima are trade marks of International Specialty Products, Inc. 1361Alps Road Wayne NJ 07470

FORMULATION 6 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 53.525Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.350 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 20.000 Neem seed oil Neem seed oil4.000 Tea tree oil Tea tree oil 1.000 Prolipid 141 ™ Emulsifier blend ofstearic acid, 6.250 behenyl alcohol, glycerol- monostearate, lecithin,C12-C16 alcohols and palmitic acid Vitamin E acetate Vitamin E acetate0.200 Ceraphyl 230 ™ Di-isopropyl adipate 7.500 PHASE C 10% w/v NaOHSodium hydroxide 0.875 Water Water 2.000 PHASE D Liquapar Optima ™Phenoxyethanol, methylparaben, 1.000 Isopropylparaben, isobutylparabenbutylparaben Total 100.000 MANUFACTURING PROCEDURE 41. In phase Acombine water, disodium EDTA and glycerol, then mix thoroughly. 42.Sprinkle Stabileze QM ™ into the pre-mixed solution with stirring atroom temperature, until uniformly dispersed, then heat to 75-80° C.while stirring, for at least 30 minutes. 43. In a separate vessel,combine ingredients of phase B; mix and heat to 75-80° C. 44. Add phaseB to phase A, homogenise for 3-5 minutes, then turn off the heat source.45. Add phase C into homogenate of phases A & B and then homogenise for3-5 minutes with no further heating. 46. Remove homogeniser andthoroughly mix while cooling to 38-40° C. 47. Add phase D to batch andmix until uniform. 48. Adjust for water loss and mix until uniform.NOTES (a) Adjustments to preservative may have to be made afterchallenge testing. (b) Small adjustments may have to be made to theStabileze QM ™ concentration to give correct viscosity depending on theapplication. (c) Small adjustments in composition will have to be made,to allow for fragrance if required. (d) Stabileze QM, Ceraphyl 230,Prolipid 141 and Liquapar Optima are trade marks of InternationalSpecialty Products, Inc. 1361 Alps Road Wayne NJ 07470

FORMULATION 7 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 53.525Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.350 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 20.000 Neem seed oil Neem seed oil4.000 Tea tree oil Tea tree oil 1.000 Prolipid 141 ™ Emulsifier blend ofstearic acid, 6.250 behenyl alcohol, glycerol- monostearate, lecithin,C12-C16 alcohols and palmitic acid Ceraphyl 140A ™ Isodecyl Oleate 7.5Vitamin E acetate Vitamin E acetate 0.200 PHASE C 10% w/v NaOH Sodiumhydroxide 0.875 Water Water 2.000 PHASE D Liquapar Optima ™Phenoxyethanol, methylparaben, 1.000 Isopropylparaben, isobutylparabenbutylparaben Total 100.000 MANUFACTURING PROCEDURE 49. In phase Acombine water, disodium EDTA and glycerol, then mix thoroughly. 50.Sprinkle Stabileze QM ™ into the pre-mixed solution with stirring atroom temperature, until uniformly dispersed, then heat to 75-80° C.while stirring, for at least 30 minutes. 51. In a separate vessel,combine ingredients of phase B; mix and heat to 75-80° C. 52. Add phaseB to phase A, homogenise for 3-5 minutes, then turn off the heat source.53. Add phase C into homogenate of phases A & B and then homogenise for3-5 minutes with no further heating. 54. Remove homogeniser andthoroughly mix while cooling to 38-40° C. 55. Add phase D to batch andmix until uniform. 56. Adjust for water loss and mix until uniform.NOTES (a) Adjustments to preservative may have to be made afterchallenge testing. (b) Small adjustments may have to be made to theStabileze QM ™ concentration to give correct viscosity depending on theapplication. (c) Small adjustments in composition will have to be made,to allow for fragrance if required. (d) Stabileze QM, Ceraphyl 140A,Prolipid 141 and Liquapar Optima are trade marks of InternationalSpecialty Products, Inc. 1361 Alps Road Wayne NJ 07470

FORMULATION 8 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 53.525Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.350 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 25.000 Prolipid 141 ™ Emulsifierblend of stearic acid, 6.250 behenyl alcohol, glycerol- monostearate,lecithin, C12-C16 alcohols and palmitic acid Ceraphyl 140 ™ IsodecylOleate 5.000 Vitamin E acetate Vitamin E acetate 0.200 PHASE C 10% w/vNaOH Sodium hydroxide 0.875 Water Water 2.000 PHASE D Liquapar Optima ™Phenoxyethanol, methylparaben, 1.000 Isopropylparaben, isobutylparabenbutylparaben Total 100.000 MANUFACTURING PROCEDURE 57. In phase Acombine water, disodium EDTA and glycerol, then mix thoroughly. 58.Sprinkle Stabileze QM ™ into the pre-mixed solution with stirring atroom temperature, until uniformly dispersed, then heat to 75-80° C.while stirring, for at least 30 minutes. 59. In a separate vessel,combine ingredients of phase B; mix and heat to 75-80° C. 60. Add phaseB to phase A, homogenise for 3-5 minutes, then turn off the heat source.61. Add phase C into homogenate of phases A & B and then homogenise for3-5 minutes with no further heating. 62. Remove homogeniser andthoroughly mix while cooling to 38-40° C. 63. Add phase D to batch andmix until uniform. 64. Adjust for water loss and mix until uniform.NOTES (a) Adjustments to preservative may have to be made afterchallenge testing. (b) Small adjustments may have to be made to theStabileze QM ™ concentration to give correct viscosity depending on theapplication. (c) Small adjustments in composition will have to be made,to allow for fragrance if required. (e) Stabileze QM, Ceraphyl 140A,Prolipid 141 and Liquapar Optima are trade marks of InternationalSpecialty Products, Inc. 1361 Alps Road Wayne NJ 07470

FORMULATION 9 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water 51.475Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100 Stabileze QM ™Poly(methyl vinyl ether/maleic 0.400 anhydride) decadiene crosspolymerPHASE B Oil Biologically-active oil 20.000 Prolipid 141 ™ Emulsifierblend of stearic acid, 5.000 behenyl alcohol, glycerol- monostearate,lecithin, C12-C16 alcohols and palmitic acid Ceraphyl 140A ™ IsodecylOleate 5.000 Vitamin E acetate Vitamin E acetate 0.200 PHASE C 10% w/vNaOH Sodium hydroxide 1.000 Water Water 2.000 PHASE D Liquapar Optima ™Phenoxyethanol, methylparaben, 1.000 Isopropylparaben, isobutylparabenbutylparaben Total 100.000 MANUFACTURING PROCEDURE 65. In phase Acombine water, disodium EDTA and glycerol, then mix thoroughly 66.Sprinkle Stabileze QM ™ into the pre-mixed solution with stirring atroom temperature, until uniformly dispersed, then heat to 75-80° C.while stirring, for at least 30 minutes. 67. In a separate vessel,combine ingredients of phase B; mix and heat to 75-80° C. 68. Add phaseB to phase A, homogenise for 3-5 minutes, then turn off the heat source.69. Add phase C into homogenate of phases A & B and then homogenise for3-5 minutes with no further heating. 70. Remove homogeniser andthoroughly mix while cooling to 38-40° C. 71. Add phase D to batch andmix until uniform. 72. Adjust for water loss and mix until uniform.NOTES (a) Adjustments to preservative may have to be made afterchallenge testing. (b) Small adjustments may have to be made to theStabileze QM ™ concentration to give correct viscosity depending on theapplication. (c) Small adjustments in composition will have to be made,to allow for fragrance if required. (d) Stabileze QM, Ceraphyl 140A,Prolipid 141 and Liquapar Optima are trade marks of InternationalSpecialty Products, Inc. 1361 Alps Road Wayne NJ 07470

FORMULATION 10 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water67.950 Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100Stabileze QM ™ Poly(methyl vinyl ether/maleic 0.500 anhydride) decadienecrosspolymer PHASE B Oil Biologically-active oil 10.000 Vitamin EVitamin E 2.000 Vitamin C Palmitate Vitamin C Palmitate 3.000 Prolipid141 ™ Emulsifier blend of stearic acid, 4.000 behenyl alcohol, glycerol-monostearate, lecithin, C12-C16 alcohols and palmitic acid Ceraphyl140A ™ Isodecyl Oleate 5.000 Vitamin E acetate Vitamin E acetate 0.200PHASE C 10% w/v NaOH Sodium hydroxide 1.250 Water Water 2.000 PHASE DLiquapar Optima ™ Phenoxyethanol, methylparaben, 1.000 Isopropylparaben,isobutylparaben butylparaben Total 100.000 MANUFACTURING PROCEDURE 73.In phase A combine water, disodium EDTA and glycerol, then mixthoroughly. 74. Sprinkle Stabileze QM ™ into the pre-mixed solution withstirring at room temperature, until uniformly dispersed, then heat to75-80° C. while stirring, for at least 30 minutes. 75. In a separatevessel, combine ingredients of phase B; mix and heat to 75-80° C. 76.Add phase B to phase A, homogenise for 3-5 minutes, then turn off theheat source. 77. Add phase C into homogenate of phases A & B and thenhomogenise for 3-5 minutes with no further heating. 78. Removehomogeniser and thoroughly mix while cooling to 38-40° C. 79. Add phaseD to batch and mix until uniform. 80. Adjust for water loss and mixuntil uniform. NOTES (a) Adjustments to preservative may have to be madeafter challenge testing. (b) Small adjustments may have to be made tothe Stabileze QM ™ concentration to give correct viscosity depending onthe application. (c) Small adjustments in composition will have to bemade, to allow for fragrance if required. (d) Stabileze QM, Ceraphyl140A, Prolipid 141 and Liquapar Optima are trade marks of InternationalSpecialty Products, Inc. 1361 Alps Road Wayne NJ 07470

FORMULATION 11 COMPOSITION DESCRIPTION (% w/w) PHASE A Water Water57.300 Glycerol Glycerol 3.000 Disodium EDTA Disodium EDTA 0.100Stabileze QM ™ Poly(methyl vinyl ether/maleic 0.400 anhydride) decadienecrosspolymer PHASE B Oil Biologically-active oil 20.000 Prolipid 141 ™Emulsifier blend of stearic acid, 5.000 behenyl alcohol, glycerol-monostearate, lecithin, C12-C16 alcohols and palmitic acid MethylSalicylate Methyl Salicylate 10.000 Vitamin E acetate Vitamin E acetate0.200 PHASE C 10% w/v NaOH Sodium hydroxide 1.000 Water Water 2.000PHASE D Liquapar Optima ™ Phenoxyethanol, methylparaben, 1.000Isopropylparaben, isobutylparaben butylparaben Total 100.000MANUFACTURING PROCEDURE 81. In phase A combine water, disodium EDTA andglycerol, then mix thoroughly 82. Sprinkle Stabileze QM ™ into thepre-mixed solution with stirring at room temperature, until uniformlydispersed, then heat to 75-80° C. while stirring, for at least 30minutes. 83. In a separate vessel, combine ingredients of phase B; mixand heat to 75-80° C. 84. Add phase B to phase A, homogenise for 3-5minutes, then turn off the heat source. 85. Add phase C into homogenateof phases A & B and then homogenise for 3-5 minutes with no furtherheating. 86. Remove homogeniser and thoroughly mix while cooling to38-40° C. 87. Add phase D to batch and mix until uniform. 88. Adjust forwater loss and mix until uniform. NOTES (a) Adjustments to preservativemay have to be made after challenge testing. (b) Small adjustments mayhave to be made to the Stabileze QM ™ concentration to give correctviscosity depending on the application. (c) Small adjustments incomposition will have to be made, to allow for fragrance if required.(d) Stabileze QM, Prolipid 141 and Liquapar Optima are trade marks ofInternational Specialty Products, Inc. 1361 Alps Road Wayne NJ 07470

B) Oral Formulations Containing Biologically Active Oils

1. Hard gel capsules (0.95 mL) made of gelatine or equivalent polymercontaining approximately 0.9 gm of oil containing 0.1% Tocopherylacetate.2. Hard gel capsules (0.95 mL) made of gelatine or equivalent polymercontaining 0.50 gm of oil dispersed in macadamia oil or equivalent.3. Soft gel capsules (100 μL up to 1.0 mL capacity) made from gelatineor equivalent polymer containing from 100 μL up to 1.0 mL of oil with0.1% anti-oxidant added if required.4. Soft gel capsules (100 μL up to 1.0 mL capacity) made of gelatine orequivalent polymer containing from 10 mg up to 1000 mg of extract fromthe oil containing if required an anti-oxidant and another oil forexample macadamia oil, soybean oil or equivalent.5. Soft gel suppositories as for 3 & 4 above.6. Syrups and lotions made from oil and extracts with the addition ofother oils such as olive, macadamia and flavours such as raspberry,strawberry, banana and with the addition of anti-oxidants if required.Dose by spoon or syringe.7. Oral dose 5.0 mL oil by spoon or syringe.

C) In Vivo Rat Model Test Procedures Used to Analyse the BiologicalActivity of Various Animal and Plant Oils and their Extracts

1. Anti-inflammatory efficacy was measured in rats developing theadjuvant-induced polyarthritis, the test agents being given eithertransdermally or orally from the time the arthritis was first expressed.Synergistic activity with low-dosed steroid was measured either in i)rats with fully established adjuvant arthritis or ii) rats with chronicpaw oedema induced by injecting 0.5 mg zymosan (in 0.2 mL saline) thenwaiting 3 hours for the acute oedema to peak (associated withhistamine/serotonin release) and measuring residual paw swelling 21-45hours later. For transdermal administration, oils were diluted with 0.15vol of cineole to facilitate skin penetration and applied once daily tothe shaved dorsal skin (6 cm²) with brief rubbing. (See Tables 1 and 2below).2. Co-arthritigenic activity was measured in Dark Agouti rats by firstdispersing finely-ground heat-killed Mycobacterium. tuberculosis in testoils (10 mg/kg) and then injecting 0.1 mL into the tailbase of femaleDark Agouti rats. Signs of arthritis were recorded on day 15. Extractsfrom emu oils were obtained by mixing equal masses of oil and methanolthen storing in a cold room or freezer at 0° C. for at least 12 hours,decanting the liquid layer, evaporating the solvent using a rotary filmevaporator. Residue remaining in the flask contains the extract. Theseextracts were first dissolved in jojoba bean oil and diluted with anequal volume of a dispersion of Mycobacterium. tuberculosis (10 mg/ml)freshly prepared injojoba bean oil. (See Table 3 below).3. Gastroprotectant activity was ascertained in a) disease stressed(untreated) polyarthritic female Dark Agouti or Wistar rats and b)normal Dark Agouti or Wistar rats which had been fasted overnight andinjected with the cholinergic drug, methacholinehydrochloride (5 mg/kgi.p.). Test materials were emulsified with 0.04% v/v Tween-20 using aVortex homogeniser, then co-dosed with a dispersion of OTC ibuprofen(NUROFEN) 55 mg/kg used as the gastrotoxin. The stomachs were removed2.5 hours later, briefly rinsed in saline and scored for number andseverity of macroscopic haemorrhagic lesions in the gastric mucosa. (SeeTables 4 and 5 below).4. Synergistic activities of emu and macadamia oils with corticosteroidsfor suppressing Zymosan-induced paw oedema in rats. A single treatmentof whole oil, or extract, plus either P=prednisone 2.5 mg/kg orD=dexamethasone 0.1 mg/kg in Tween-20 was administered orally 3 hoursafter injecting 0.5 mg zymosan into each rear paw. Data are the relativereduction in paw swelling compared with controls treated with olive oilonly, expressed as percentage inhibition. (see Table 6 below).5. For in vitro tests, oils were processed to remove the bulk of thetriglycerides using solvent extraction at low temperature or solid-phaseextraction in accordance with normal laboratory procedures. For in vivotests, the oils were filtered at 22° C. to remove solids, varying from5-45% by weight. Exceptionally stiff samples were diluted either with0.1 volume n-octanol or up to 0.5 volumes isopropyl myristate to help‘liquefy’ them, these solvents being inert vehicles for the assaysdescribed.Note: All Mycobacterium. tuberculosis used is finely ground and heatkilled prior to use.

Results

1. Wistar rats were injected with 0.8 mg Mycobacterium. tuberculosis in0.1 mL squalene in tailbase (day 0). Treatments with biologically activeoils were given eitherA) transdermally on days 10-13 only (4 rats/gp) or B) orally on days15-17 together with prednisone (2.5 mg/kg, 3 rats/gp)

The changes in arthritic signs are shown below. An increase in weight isgood, a decrease indicates possible toxicity. The lower the arthriticscore, the better:

Note: For information on oils refer to chromatogram number and table 6which contains process conditions.

Process conditions and chromatograph for each sample in Tables 1 to 8are summarised in Table 11. The corresponding chromatograms are numbered1 to 32. As an example, emu-type 2 oil in Table 3 was obtained accordingto the process conditions set out in line 4 of Table 11 (sample codeType 2) and the biological activity of the sample is shown inchromatogram 4.

TABLE 1 A. Treatment Mean Changes in Arthritic (transdermal) Signs (Days10→14) Dose/ Rear paw Forepaw ΔWeight Arthritis Sample kg/day thicknessinflammation gm Score Olive 2.0 mL 0.89 mm 2+   +05 2+   Oil - ControlEmu-A 0.5 mL 0.15 mm 0.5+ +14 0.5+ Chrom. 1. Emu-C 1.0 mL 0.37 mm 1.4++18 1+   Chrom. 2. Emu-Kalaya 0.5 mL 0.08 mm −0.2+   +05 0.2+ Chrom. 3.

TABLE 2 B. Treatment (Oral) Mean Changes in Arthritic Signs (Days 15→18)Dose/ Rear paw Forepaw ΔWeight Arthritis Sample kg/day thickness Tailthickness inflammation gm Score Olive Oil 2.0 mL    0.0 mm +0.42 mm 0.6++02 0.5+ (OO) only - control Prednisone with: Olive Oil 2.0 mL −0.05 mm  0.15 mm 0.3+ +03 0 Emu-A 2.0 mL −0.59 mm −0.82 mm −0.8+ 0 −0.9+ Chrom.1 Emu-C 2.0 mL +0.13 mm −0.14 mm 0.8+ −01 0.3+ Chrom. 2

Table 3: Arthritigenic Activity of Some Emu/Other Oils

Oils were admixed with finely ground heat-killed M. tuberculosis (10mg/mL) and 0.1 mL injected into the tail base of female Dark Agoutirats.Dispersions with jojoba bean oil contained a final concentration of only5 mg/mL M. tuberculosis.Signs of arthritis were scored on day 15 for groups of 3 rats.

Mean values for Rear paw Arthritis Test Oil swelling ΔWeight Score OliveOil 1.22 mm +01 2.3+ Lard Oil (pig) 1.03 +14 2.3+ Emu-A Chrom. 1 0.23+08 0.5+ Emu-C Chrom. 2 0.73 −10 2+   Emu-Kalaya Chrom. 3 0.08 +21 0.7+Emu-Type 2 Chrom. 4 0.20 +12 0.5+ Jojoba Bean 1.24 +02 2.8+ with extr.Emu-A (5 mg/rat) 0.23 +11 0.7+ with extr. Emu-C (5 mg/rat) 0.92 +07 1.5+with extr. Emu-Ka (5 mg/rat) 0.08 +11 0.3+ with extr. Emu-Ka (10 mg/rat)0.09 +20 0.5+

Tables 4/5: Gastroprotective Activity of Some Emu Oils in Rats

Gastro-irritant=55 mg/kg ibuprofen given orally to animals fastedovernight, together with test emulsions=0.4 mL oil/kg prepared with0.04% v/v Tween-20 along with or without methacholine given i.p.

A. In disease-stressed female Wistar or Dark Agouti rats with fullydeveloped polyarthritis (on or after day 15), without methacholine.

TABLE 4 Gastric lesion indices in Treatment Wistar rats n = 3/gp DarkAgouti rats n = 4/gp Tween-20 only 32 44 Emu-A Chrom. 1 07 22 Emu-CChrom. 2 21 51 Emu-Kalaya Chrom. 3 not tested 27B. In normal rats stimulated with Beta-methacholine (5 mg/kg in Wistarrats or 8 mg/kg in Dark Agouti rats).

TABLE 5 Gastric lesion indices in Dark Agouti Treatment Wistar rats n =3/gp rats n = 3/gp Tween-20 only 17 52 Emu-A Chrom. 1 05 17 Emu-C Chrom.2 19 39 Emu-Kalaya Chrom. 3 05 23 Olive oil (OO) 19 31 Extr. A = 50mg/kg in OO 06 10Table 6: Synergistic Activities of Emu and Macadamia Oils withCorticosteroids for Suppressing Zymosan-Induced Paw Oedema in Rats.

A single treatment of whole oil, or extract, plus either P=prednisone2.5 mg/kg or D=dexamethasone 0.1 mg/kg in Tween-20 was administeredorally 3 hours after injecting 0.5 mg zymosan into each rear paw. Dataare the relative reduction in paw swelling compared with controlstreated with olive oil only, expressed as percentage inhibition.

Dose/ Wistar rats Dark Agouti rats Treatment kg Day 1 Day 2 Day 1 Day 2P + olive oil (OO) 2 mL 4% 1% 15% 2% P + Emu - A Chrom. 1 2 mL 52 81 5663 0.5 43 52 P + Emu - C Chrom. 2 2 mL −05 −16 −14 −12 P + Lyprinol inOO 20 mg 57 31 65 66 D + olive oil (OO) 2 mL 05 0 D + Emu - 2 mL 77 40 AChrom. 1 D + Emu - 2 mL 22 −15 C Chrom. 2 D + Lyprinol in OO 20 mg 56 50P + Olive oil (OO) 2.0 mL 0 P + Macadamia - 19 1.6 mL 1 Chrom. 30 P +Macadamia - 20 1.6 mL 41 Chrom. 31 P + Oleic acid (90%) 2.0 mL −11 P +Isostearic acid 2.0 mL 39 (comm.) P + Lyprinol in OO 20 mg 46D) In-vitro Lipoxygenase assays of Several Different Oil Samples

Neutrophil 5-Lipoxygenase Pathway Overview

Arachidonic acid is converted into eicosanoids (or prostanoids) by twomajor pathways, the 5-lipoxygenase pathway, which leads to the formationof leukotrienes, and the cyclo-oxygenase pathway which leads to theformation of prostaglandins and thromboxanes. Some, but not all, of theproducts of both of these pathways have potent pro-inflammatoryproperties. For example, LTB₄ is a very potent chemotactic agent, andits peptido-metabolites, LTC₄, LTD₄ and LTE₄, which were originallyknown as “slow reacting substance of anaphylaxis” or SRS-A, are potentbronchoconstrictor agents.

Many of the currently used anti-inflammatory drugs, in particular thenon-steroidal anti-inflammatory drugs (NSAIDS), function via theinhibition of the cyclo-oxygenase pathway. More recently, considerableeffort around the world has focused on the development of inhibitors ofthe lipoxygenase pathway, or of dual inhibitors that block bothpathways.

The principal steps of the 5-lipoxygenase pathway of these cells isshown in FIG. 3. In this pathway, arachidonic acid (AA), from membranephospholipids, is released via the action of phospholipase A₂ (PLA₂).This AA is then substrate for the first enzyme in the pathway—5lipoxygenase, which converts it to 5-hydroperoxyeicosatetraenoic acid(5-HPETE). 5-HPETE is then converted enzymatically to either5-hydroxyeicosa-tetraenoic acid (5-HETE) by glutathione peroxidase, orto leukotriene A₄ (LTA₄) by LTA₄ synthase. LTA₄ is then converted eithernon-enzymatically to the all trans isomers of LTB₄, or hydrolysed byLTA₄ hydrolase to leukotriene B₄ (LTB₄). Human PMN do not significantlymetabolise LTB₄ any further, although other cells, such as eosinophilsconvert it to the potent vasoconstrictor peptido-leukotrienes, SRS-A.

The Lipoxygenase Pathways (Leukotrienes AND HETE) Assay

The HPLC assay readily quantifies 5-HETE, 12-HETE, LTB₄, and the two alltrans-isomers of LTB-₄, and thus gives quantitative data on the relativeactivities of the enzymes in the 5-lipoxygenase of neutrophils, as wellas data on the 12-lipoxygenase pathway of platelets. Thus it is an idealsystem to test potential inhibitors of these pathways.

The effects of inhibitory compounds may be tested on isolated human PMNand platelets, in which the pathways are activated by treating the PMNor platelets with arachidonic acid and the calcium ionophore A23187. Theaddition of arachidonic acid eliminates the PLA-₂ step, and provideshigh levels of substrate for the pathway. Furthermore, such activationis known to maximally drive the pathway to produce the greatestsynthesis of all the metabolites, and thus is also the least sensitiveto inhibition. Hence, compounds that do inhibit the pathway activated inthis fashion are potentially potent inhibitors.

Preparation of Test Samples

All samples were dissolved in ethanol to give stock solutions of 10mg/mL. Two further dilutions of each stock solution were made in ethanolat 5 and 1 mg/mL, making 10 test samples in all.

10 μL of each of the diluted stocks was added to 1000 μL of PMNsuspension in Hank's Buffer, to give final test concentrations of 5, 10and 50 ug/mL as required for the analysis.

Preparation of Human Neutrophils (PMN)

1. Up to 100 mL of blood was taken from a normal volunteer andanticoagulated with EDTA. Two mL of 4.5% EDTA in water was mixed witheach 10 mL of blood.2. A further 2 mL of 6.0% Dextran T500 was added to each of the 12 mLmixtures in 1, and placed in a water bath at 37° C., to sediment the redblood cells.3. Following sedimentation in 2, the supernatant was carefully laid over5 mL of Percoll, density 1.070. This was then spun at 500 g for 35 mins.4. All the cells (PMN and remaining RBC) below the Percoll interfacewere removed with a plastic pipette and diluted at least 3-fold withCa²⁺/Mg²⁺-free Dulbecco's phosphate buffer, and centrifuged at 600 g for10 mins.5. Following 4, the supernatant was carefully aspirated, and the pelletgently mixed with 1 mL of Ca²⁺/Mg²⁺-free Dulbecco's phosphate buffer byaspiration/deaspiration into a 1 ml plastic disposable pipette. Afurther 40 mL of Ca²⁺/Mg²⁺-free Dulbecco's phosphate buffer was thenadded and mixed by inversion. The cell suspension was then centrifugedat 600 g for 10 mins.6. Following centrifugation, the supernatant was removed and the PMNpellet lysed with 10 mL of a 0.2% cold sodium chloride solution for 20secs, followed by the addition of 10 mL of a 1.6% cold sodium chloridesolution, and centrifuged at 600 g for 10 mins.7. Following 6, the PMN pellet was vigorously mixed with 1 mL of Hank'sbuffer by rapid aspiration/deaspiration into a 1 mL plastic disposablepipette, and then finally suspended in Hank's buffer at 2.4×10⁶ PMN/ml(as measured using a Coulter counter), in preparation for theleukotriene assay.

Activation of Leukotriene Pathway

1. 1 mL of PMN suspension (2.4×10⁶ PMN/ml) was transferred to a 13 mLglass tube (chromic acid washed) and placed in a water bath at 37° C.for 5 min prewarming.2. Following prewarming, at time zero, 10 μL of each test compound inmethanol (or equivalent volumes of methanol as control) was added toquadruplicate tubes over a 20 sec period.3. At 5 min, 5 □L of 2 mM arachidonic acid (10 μM final) was added (4tubes/20 secs).4. At 10 min, 5 □L of 1 mM calcium ionophore (A23187) (5 μM final) wasadded (4 tubes/20 secs).5. At 15 min the reaction was terminated by the addition of 100 μL 100mM citric acid. This lowers the pH of the aqueous phase to less than 3,which is necessary for the extraction of the leukotrienes into theorganic phase.6. The pH of several samples was checked to ensure pH<3.0. (This isimportant).7. 40 ng Prostaglandin B2 and 166 ng 15-HETE were added to each tube asthe internal standard for LTB4 and 5-HETE respectively, and samples weremixed.8. For Standard Curves LTB4 [1 ng/μl] (for a standard curve in the range0-50 ng) and 5-HETE [5 ng/μL] (standard curve range 0-250 ng) were addedto tubes containing 1 mL PMN, 100 μL of 100 mM citric acid and 40 ngPGB₂ and 166 ng 15-HETE.9. All tubes were vortexed.10. 5 mL chloroform/methanol (7:3) was added and the tubes vortexedvigorously for 30 secs, then centrifuged for 10 min at 2000 rpm.11. Approx. 3.5 mL of the lower chloroform layer (containing theextracted leukotrienes and hydroxy acids (HETES), as well as theinternal standards) was transferred to a 3 mL borosilicate glass tubeand the chloroform evaporated in a Savant centrifugal evaporator, undervacuum, at room temperature.12. The samples were reconstituted in 100 μL of the LTB₄ mobile phase,vortexed and transferred to Waters low volume inserts for injection(usually <25 μl).13. The HPLC was setup for the LTB₄ conditions, and all the samplesassayed for LTB₄ and the all trans isomers of LTB₄.

HPLC ASSAY FOR LEUKOTRIENES and HYDROXY ACIDS Mobile Phases LTB4 Assay:70% Methanol/30% H₂O/0.08% Acetic Acid (pH adjusted to 6.2 with ammoniumhydroxide). 5-HETE Assay: 80% Methanol/20% H₂O/0.08% Acetic Acid (pHadjusted to 6.2 with ammonium hydroxide). HPLC Conditions Wavelength:270 nm (LTB₄), 234 nm (5-HETE) Analysis: Water's Millennium Flow Rate: 1mL/min Column and Guard Pak: C₁₈ Nova Pak LTB₄ ASSAY Retention TimesProstaglandin B₂ - 4.6 min 6-trans-leukotriene B₄ - 6.6 min6-trans-epi-leukotriene B₄ - 7.4 min Leukotriene B₄ - 8.7 minFull Chemical Names of LTB₄ and its 6-trans IsomersLeukotriene B₄: (5S,12R)-Dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoicacid.6-trans-Leukotriene B₄: (5S,12R)-Dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid.6-trans-12-epi-LeukotrieneB₄:(5S,12S)-Dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid

5-Hete Assay

Retention Times

-   -   15-HETE—6.3 min    -   5-HETE—8.5 min

Full Chemical Names

-   -   15-HETE: 15(S)-Hydroxy-(Z,Z,Z,E)-5,8,11,13-eicosatetraenoic acid    -   5-HETE: 5(S)-Hydroxy-(E,Z,Z,Z)-6,8,11,14-eicosatetraenoic acid

TABLE 7 Effects of Emu Oil Methanol Extracts and pure 12-methyltetradecanoic acid (12-MTA) on Leukotriene Synthesis (Expressed as apercentage of the methanol control) Test Material Dilution Isomer 1Isomer 2 LTB₄ 5-HETE Control 100 ± 22 100 ± 22 100 ± 4  100 ± 4 Emu-Type 50 μg/mL 0 0 11 ± 8 10 ± 5 2 Chrom. 4 Emu-Type 10 μg/mL  88 ±12  71 ± 11 74 ± 6  76 ± 16 2 Chrom. 4 Emu-Type  5 μg/ml  94 ± 12  99 ±13  84 ± 10 102 ± 2  2 Chrom. 4 Emu-Type 50 μg/mL 23 ± 3 20 ± 4  17 ± 11 16 ± 11 2* Chrom. 4 Emu-Type 10 μg/mL 78 ± 6 76 ± 7 80 ± 4 78 ± 7 2*Chrom. 4 Emu-Type  5 μg/mL 95 ± 9 96 ± 8 88 ± 2 90 ± 7 2* Chrom. 4Emu-Type-B 50 μg/mL 0 0 0  4 ± 2 Chrom. 24 Emu-Type-B 10 μg/mL  82 ± 14 86 ± 14 88 ± 9 66 ± 9 Chrom. 24 Emu-Type-B  5 μg/mL 110 ± 11 137 ± 9 95 ± 8 92 ± 1 Chrom. 24 12-MTA 50 μg/mL 0 0 0 0 12-MTA 20 μg/mL 0 0 0  2± 4 12-MTA 10 μg/mL  59 ± 14  68 ± 14  70 ± 17  46 ± 11 12-MTA  5 μg/mL101 ± 12 97 ± 9 101 ± 12 65 ± 3 12-MTA  2 μg/mL  73 ± 13  60 ± 17 84 ± 555 ± 9 *Prior to extraction the original oil sample was treated bypassing nitrogen gas at high flow rate through oil heated at 135° C. fortwo hours with rapid stirring, to remove volatile compounds.

TABLE 8 Effects of Fatty acid methyl ester of emu oil, methanol extractsof emu and ostrich oil produced by fungal inoculation and incubation onLeukotriene Synthesis(Expressed as a percentage of the methanol control)Test Material Dilution Isomer 1 Isomer 2 LTB₄ 5-HETE Control 100 ± 9 100 ± 10 100 ± 6  100 ± 5  Fatty acid methyl ester of emu oil* 50 μg/mL  7 ± 0.5 0  8 ± 1  6 ± 2 Fatty acid methyl ester of emu oil* 10 μg/mL103 ± 9  103 ± 8  95 ± 6 105 ± 4  Emu oil-WB methanol extract 50 μg/mL  4 ± 0.5 0 0 23 ± 4 Chrom. 21 Emu oil-WB methanol extract 10 μg/mL 61 ±2  65 ± 2 80 ± 3 85 ± 3 Chrom. 21 Ostrich oil methanol extract 50 μg/mL9 ± 2 14 ± 3 22 ± 6 21 ± 8 Chrom. 22 Ostrich oil methanol extract 10μg/mL 56 ± 12  74 ± 17  65 ± 11  83 ± 13 Chrom. 22 *Fatty acid methylester (FAME) of emu oil sample A, Chromatogram 1.

Results and Discussion

The data shows that all three samples in table were potent inhibitors ofthe 5-LOX pathway. The data are compared to 12-methyl tetradecanoic acidwhich showed 100% inhibition as low as 20 μg/mL.

With respect to samples in Table 8, FAME sample was the least effective,where as Emu oil-WB and Ostrich oil samples are approximately the sameas the three samples in Table 7. This confirms that the process for theproduction of biologically-active oils may be reproduced using differentlipid substrates etc.

E) In-vitro Prostaglandin PGE₂ (COX Pathways) Assay Oil Samples

This assay was performed using Cayman chemicals Prostaglandin E₂ EIAKit-Monoclonal, according to kit protocol. Each sample was assayed atthree dilutions in duplicate. As can be seen from Table 9 the inhibitionof the PGE₂ response to aspirin (50 μM) was around 72.6% of the controlvalue. In particular the two samples produced dose dependent inhibitionof secreted PGE₂ from the mouse fibroblast cell line equivalent to orbetter than aspirin under the test conditions.

TABLE 9 Percent Inhibition of Secreted PGE₂ from 3T3 cells exposed toOil Extracts Concentration Sample □g/mL % Control DMSO + A23187 100Aspirin 50 □M 72.6 Emu Oil Extract - P3 Chrom. 3 100 52 Emu OilExtract - P3 Chrom. 3 20 59 Emu Oil Extract - P3 Chrom. 3 4 84.8Lyprinol - P10 100 60.9 Lyprinol - P10 20 83.6 Lyprinol - P10 4 84.6

F) Examples of Therapeutic Activity

1) Patient suffering from ulcerative colitis for seven years hadexperienced chronic diarrhoea and daily rectal bleeding. Patientingested 5 mL of fungi-derived biologically-active emu oil twice daily.All anal bleeding associated with the ulcerative colitis disappearedafter 2 months. This patient has also responded to fungi-derivedbiologically-active ostrich oil.

2) Patient suffering chronic pain from intestinal and duodenal ulcersingested 5 mL of fungi-derived biologically-active emu oil twice dailyand pain has ceased. Patient also observed that his unstable diabetesbecame more responsive to the insulin resulting in a reduction of dosagerequired.

3) Patient diagnosed with Crohn's disease 5 years ago and was sufferingongoing abdominal pain, diarrhoea or constipation, rectal bleeding, coldsweats and lethargy. After weeks ingesting 5 mL of fungi derivedbiologically-active emu oil twice daily. The patient's Crohn's diseaseis in remission (this has been confirmed by medical tests) with nofurther abdominal pains, diarrhoea or constipation.

4) Patient diagnosed with breast cancer, had lumpectomy, radiation andchemotherapy. Topical application of cream produced from fungi-derivedbiologically-active oil applied three times daily which reduced pain andinflammation in the breast.

5) Patient is a 54 year old Caucasian male with a 10 year history ofmild to moderate asthma, which was controlled with 400 ug twice daily ofbeclomethasone and either salbutamol or turbutamine bronchodilatorinhalers where needed. This was inadequate to control viral inducedasthma following winter infections where oral prednisone at 5 mg/day wasrequired to reduce chronic wheezing and coughing to an acceptable level.The patient was administered 8 g/day of fungi-derivedbiologically-active emu oil in divided doses-4 g morning and night.Within 3 weeks all asthma symptoms reduced, and improvement continuedfollowing cessation of aerosol steroids. After 2 months on the oil, thepatient controlled all symptoms of asthma with 4 g/day of the oil and noother medication. In addition, the administration of the oil has reducedthe need for LOSEC to be taken for the patient's gastric reflux.

6) Patient is a 62 year old female with a 51 year history of chronicasthma (Classified as chronic airways limited), which was controlled by10-50 mg/day of oral prednisone, 900 mg/day of neulin, plus frequent useof ventolin/atrovent puffers and nebules was required to control chronicwheezing and coughing to an acceptable level. The patient wasadministered 6 ml of fungi-derived biologically-active emu oil individed doses, 3 mL morning and night. The patient has been on this doseof oil for 11 months and this has virtually eliminated the wheezingnoise, reduced the level of coughing, plus the level of prednisone hasbeen reduced to 5 mg/day. Also use of puffers and nebules has beenreduced. No longer needs to take Losec for gastric reflux. By increasingthe emu oil to 9 mL/day, three 3 mL doses/day with a slight increase inprednisone to 10 mg/day any asthma attacks can be controlled (the oilsynergistically increases activity of prednisone, as confirmed by a ratmodel). Her daily life style has been greatly improved since commencinguse of this oil.

7) Patient is an Eurasian Male patient 23 years of age, diagnosed bycolonoscopy, and is prescribed 40 mg prednisone daily reduced 5 mg everytwo weeks, 2 grams of mesalamine daily. 6 weeks later after bloodresults received, the patient is hospitalised and administered 7 days ofhydrocortisone IV, lost 4 kg in weight, heads of hip bones began to dieoff as a resulting side effect of the medication. The patient isprescribed post-hospital medication of:

100 mg Imuran daily (intended four year treatment)50 mg of prednisone daily (reducing 5 mg every two weeks).The patient then began taking 5 ml of oil (batch 365) three times a day,and ceased all other medication within one month from starting on theoil. His medical problems continued to reduce in severity. Three monthsafter starting on the oil the patient was instructed to take extract ofoil (batch 365), two teaspoons daily, and has experienced no chronicsymptoms in four months. The patient's health continues to improve(digestive system, stamina, fitness etc), with a weight gain of 5 kg.Cumulative C Reactive Protein reduced over a period of six months from65.1 to 5.3 mg/L, Range (0.0-5.0) mg/L. The patient's results aresummarised in Table 10 below.

CRP and ESR Results

TABLE 10 C Reactive Protein (High Sensitivity) ESR Date Range (0.0-5.0)mg/L Range (0-20) mm/hr 07/08/03 65.1 9/09/03 25.9 21/10/03 33.509/12/03 5.2 4 10/02/03 4.9 3 13/04/03 5.3 1

TABLE 11 Chromatogram Sample Lipid Fungal Humid- Temperature Time NumberCode Substrate Mixture ity (° C.) (days) 1 A emu Rhodotorulamucilaginosa, Cryptococcus albidus, Trichosporon pullulans, High 10-12128 Mucor spp, Epicoccum purpurescens, Rhizopus stolonifer, Penicilliumchrysogenum, Nigrospora sphaerica, Chaetomium globosum, Alternariaalternata 2 C emu No fungi High 10-12 128 3 Kalaya emu Rhodotorulamucilaginosa, Cryptococcus albidus, Trichosporon pullulans, Medium 10-15128 Mucor spp, Epicoccum purpurescens, Rhizopus stolonifer, Penicilliumchrysogenum, Nigrospora sphaerica, Chaetomium globosum 4 Type 2 emuRhodotorula mucilaginosa, Cryptococcus albidus, Trichosporon pullulans,High 15-20 128 Mucor spp, Epicoccum purpurescens, Rhizopus stolonifer,Penicillium chrysogenum, Nigrospora sphaerica, Chaetomium globosum 5E113 emu Mucor BB14 High 20 21 6 E115 emu Mucor BB18 High 20 21 7 E108emu Penicillium chrysogenum High 20 21 8 E109 emu Rhodotorulamucilaginosa High 20 21 9 E110 emu Cryptococcus albidus High 20 21 10E111 emu Trichosporon pullulans High 20 21 11 E112 emu Trichosporonpullulans, Rhodotorula mucilaginosa, Cryptococcus albidus High 20 21 12E116 emu Mucor Black High 20 21 13 E117 emu All Mucor spp High 20 21 14E118 emu Trichosporon pullulans, Rhodotorula mucilaginosa, Cryptococcusalbidus, High 20 21 Penicillium chrysogenum, Mucor BB14, Mucor BB16,Mucor BB18, Mucor Black 15 E104 emu Mucor BB14, Mucor BB16, Mucor BB18,Mucor Black High 20 21 16 E119 emu Nil High 20 21 17 L17 lamb MucorBlack High 20 24 18 E90 lamb Penicillium chrysogenum, Mucor BB12, MucorBB13, Mucor BB15, Low to 20 7 Mucor Black medium 19 E60 macadamiaCryptococcus albidus Low 20 14 20 E80 macadamia Cryptococcus albidus Low20 21 21 TLWB1 emu Rhodotorula mucilaginosa, Cryptococcus albidus,Trichosporon pullulans, Medium 10-12 14 Mucor spp, Epicoccumpurpurescens, Rhizopus stolonifer, Penicillium chrysogenum, Nigrosporasphaerica, Chaetomium globosum, Alternaria alternata 22 TLOSTF ostrichRhodotorula mucilaginosa, Cryptococcus albidus, Trichosporon pullulans,Medium 10-12 14 Mucor spp, Epicoccum purpurescens, Rhizopus stolonifer,Penicillium chrysogenum, Nigrospora sphaerica, Chaetomium globosum,Alternaria alternata 23 ZB2 beef Trichosporon pullulans, Rhodotorulamucilaginosa, Cryptococcus albidus, Low 20 63 Penicillium chrysogenum,Mucor BB14, Mucor BB16, Mucor BB18, Mucor Black 24 Type-B emuRhodotorula mucilaginosa, Cryptococcus albidus, Trichosporon pullulans,Medium 10-12 28 Mucor spp, Epicoccum purpurescens, Rhizopus stolonifer,Penicillium chrysogenum, Nigrospora sphaerica, Chaetomium globosum,Alternaria alternata 25 M11 macadamia Chaetomium sp medium 20 35 26 M14macadamia Penicillium, Chaetomium, Absidia and Mucoraceous fungi, mixedthrough medium 20 35 crushed nuts 27 M14A macadamia Absidia sp medium 2035 28 M15 macadamia Penicillium, Chaetomium, Absidia and Mucoraceousfungi, fungi medium 20 35 inoculated on surface of crushed nuts. 29 M16macadamia Mucoraceous fungus medium 20 35 30 M19 macadamia Penicillium,Chaetomium, Absidia and Mucoraceous fungi, fungi high 10 35 inoculatedon surface of crushed nuts 31 M20 macadamia Penicillium janczewskimedium 20 28 32 M21 macadamia Penicillium sclerotiorum medium 20 28

1. A solid state process for the production of fats or oils and/or theirextracts containing biologically-active chemical compounds from a lipidsubstrate, the process comprising: a) Inoculation of a lipid substratewith a fungal mixture having enzymatic activity, said fungal mixturebeing derived from said substrate, b) Incubating the inoculatedsubstrate for a period of between about 7-120 days at a temperature ofbetween about 4-35° C., at a humidity of between about 75-100%, suchthat said fungal mixture metabolises/transforms the lipid substrate intosaid fats or oils and/or their extracts containing biologically-activechemical compounds, and c) Processing said incubated substrate mixtureto obtain a biologically active fat or oil.
 2. A solid state process forthe production of fats or oils and/or their extracts containingbiologically-active chemical compounds from a lipid substrate, theprocess comprising: a) Incubating the lipid substrate without anyinoculation of a fungal mixture, for a period of between about 7-120days at a temperature of between about 4-35° C. at a humidity of betweenabout 75-100%, such that the lipid substrate transforms into said fatsor oils and/or their extracts containing biologically-active chemicalcompounds, and b) Processing said incubated substrate mixture to obtaina biologically active fat or oil.
 3. The process according to claim 1wherein in step b), the period of incubation is between about 7 to 56days, at a temperature of between about 5-20° C. and at a humidity ofbetween about 80-100%.
 4. The process according to claim 2 wherein instep a), the period of incubation is between about 7 to 56 days, at atemperature of between about 5-20° C. and at a humidity of between about80-100%.
 5. The process according to claim 1 wherein in step c), thelipid substrate is animal-derived and said metabolised/transformedinoculated lipid substrate is rendered to obtain a biologically activeoil.
 6. The process according to claim 2 wherein in step b), the lipidsubstrate is animal-derived and said metabolised/transformed lipidsubstrate is rendered to obtain a biologically active oil.
 7. Theprocess according to claim 1, wherein in step c), the lipid substrate isplant or seed derived, and said biologically active oil is obtained bycold pressing or solvent extraction of said inoculated substratemixture.
 8. The process according to claim 2, wherein in step b), thelipid substrate is plant or seed derived, and said biologically activeoil is obtained by cold pressing or solvent extraction of said substratemixture.
 9. The process according to claim 1 wherein a concentratedextract of a biologically active oil is prepared by solvent extractionof the obtained biologically active oil, using, methanol at lowtemperature.
 10. A biologically active fat or oil and extracts thereofproduced according to the process of claim
 1. 11. A method of treatingor preventing a disease or condition in a human or animal patient, whichcomprises administration to the patient of an effective amount of abiologically active fat or oil or extract thereof, produced according tothe process of claim
 1. 12. The method according to claim 11 whereinsaid administration is oral, transdermal or subcutaneous.
 13. The methodaccording to claim 11, wherein said condition is an inflammatorycondition including musculo-skeletal disorders such as arthritis,rheumatoid arthritis and osteoarthritis.
 14. The method according toclaim 11, wherein said disease or condition is a gastrointestinaldisorder, including inflammatory bowel diseases such as Crohn's, diseaseand ulcerative colitis, gastric ulcers, gastric reflux and pancreatitis.15. The method according to claim 11, wherein said disease is arespiratory disease, including asthma, and chronic obstructive pulmonarydisease (COPD).
 16. The method according to claim 11, wherein saiddisease is a cardiovascular disease, including atherosclerosis, coronaryartery disease and hypertension.
 17. The method according to claim 11,wherein said disease is a skin disease, including dermatitis, psoriasisand atopic eczema.
 18. The method according to claim 11, wherein saiddisease or condition is cancer, including bowel cancer, skin cancer,breast cancer, prostate cancer and sarcoidosis and non-solid tumourssuch as Hodgkin's lymphoma.
 19. The method according to claim 11,wherein said disease or condition includes leukemia, diabetes, allergies(e.g., otitis media, ocular allergy, uveitis), dysmenorrhoea, kidneydiseases (e.g., glomerulonephritis, nephrotic syndrome), benign prostatehyperplasia, septic shock.
 20. The method according to claim 11, whereinpathogenesis of said disease or condition involves activation of thelipoxygenase (LOX) pathways, including the 5-, 12- and 15-LOX pathways.21. The method according to claim 11, wherein pathogenesis of saiddisease or condition involves activation of the of cyclo-oxygenase (COX)pathways, including COX-1 and COX-2 pathways.
 22. The method accordingto claim 11, wherein pathogenesis of said disease or condition ischaracterised by an increase in blood C-reactive protein levels.
 23. Themethod according to claim 11, wherein said biologically active fat oroil or extract thereof is topically administered in the form of apharmaceutical cream, prepared with one or more acceptable carriers ordiluents.
 24. Use of a biologically active fat or oil and extractsthereof as claimed in claim 10 in the manufacture of a medicament forthe treatment of an inflammatory disease or condition in a human oranimal.
 25. The use according to claim 24 where said biologically activefat or oil inhibits the lipoxygenase (LOX) pathways, including the 5-,12- and 15-LOX pathways.